Pharmaceutical composition which is stable during storage and contains a thymus extract

ABSTRACT

The invention concerns a process for preparing a pharmaceutical composition of thymus factors which displays immunomodulating and anti-inflammatory properties and is stable during storage. The process comprises the extraction of a homogenized thymus tissue and subsequent repeated ultrafiltration via a filter membrane having an exclusion volume of 30 kD. The resultant first ultrafiltrate is then subjected to a second ultrafiltration process on a membrane having an exclusion volume of 3 kD, the retentate being used. A composition prepared in this way, in contrast to the first ultrafiltrate obtained on leukocyte cultures stimulated by phytohaemagglutinin, displays a great increase in the production of anti-inflammatory IL-10 and a reduction in the formation of inflammation-promoting IL-2 and IFN-gamma.

The invention concerns a pharmaceutical composition, which is stableduring storage and which can be obtained from a thymus extract and hasimmunomodulating and inflammation-inhibiting properties.

The production of pharmaceutical preparations from the thymus is knownand described from many aspects in the literature. Such extracts containso-called thymus factors, which comprise peptides produced from thymus.Their structure and function has still not been fully clarified in manycases. However, it is known that thymus factors cause the maturation ofT lymphocytes as well as their proliferation by lymph tissue. Inaddition, it is also known that they play a basic role in the rejectionof transplanted tissue as well as in autoimmune diseases. They are thusthe subject of intensive investigations.

Thymus factors or peptides are generally called thymosines, of whichthymostimulin and thymopoietin, thymosine-α-1, thymosine-β-4 haveperhaps been best investigated. It is known, e.g., that thymosine-α-1and thymosine-β-4 reinforce the antigen presentation to macrophages, forwhich reason they are also used for the treatment of autoimmune diseasesand, among other applications, also in defects in the immune system,such as cancer and allergies.

The preparation and extraction of thymus peptides is known anddescribed, for example, by T. L. K. Low and A. L. Goldstein in "Methodsin Enzymology", Vol. 116, p. 213 (1985). In addition, these [products]may also be obtained from sanorell pharma GmbH and Co., Rechtmurgstr.27, D-72270 Baiersbronn, e.g., under the name Thymosand®.

The storage of aqueous thymus extracts obtained by extraction, however,is limited. Thus, attempts have also been made to synthetically producepharmaceutically active partial peptides, such as, e.g., thepentapeptide of amino acids 32 to 36 of thymopoietin as well as itsanalogs (Justus Liebigs Ann. Chem. 1990, 245-247).

The invention thus has the object of making available a pharmaceuticallyusable mixture of thymus factors, which is stable during storage andwhich has a pronounced anti-inflammatory effect as well as animmunomodulating action.

This object is now achieved according to the invention in that a thymustissue homogenized in a way known in and of itself is extracted by meansof an aqueous solution, the solid components are separated from theextract and this extract is filtered through an ultrafiltration membranewith an exclusion volume of 30 kD. The process according to theinvention is now characterized by the fact that the 30 kD ultrafiltratethat is obtained is subjected to at least one additional filtration onan ultrafilter with an exclusion volume of 3 kD and the retentate isused.

It has been shown surprisingly that a retentate is obtained, which isobviously free of destabilizing factors and which retains itsimmunomodulating properties, but in addition shows pronouncedanti-inflammatory or inflammation-inhibiting properties, by means of anultrafiltration on a membrane with an exclusion volume of 3000 daltons.

The thymus cells required for the process of the invention are takenfrom young calves and are rapidly further processed after removal. Thepreparation of homogenized tissue is known to the person skilled in theart and can be produced, for example, by means of commercially availablehomogenizers and/or ultrasound treatment. The homogenate is preferablysubjected to an autodigestion for at least 10 hours, appropriately atleast 20 hours at 2-6° C., usually at 3-5° C. The extraction of thehomogenate is conducted by means of water or an aqueous solution, whichcan be buffered as needed in the neutral or biologically common pHrange. The extract thus obtained still contains solid components, whichare separated, for example, by means of centrifuging or filteringthrough filters with appropriately large pores (2.0 μm, 0.8 μm, 0.2 μm).If need be, additional separation or purification steps can be conductedwith ammonium sulfate and also other treatments, e.g., with phenol, maybe conducted, insofar as these are necessary.

The extract free from the solid components is then subjected to a firstultrafiltration on a membrane with an exclusion volume of 30 kD, wherebypreferably hydrophilic membranes, e.g., cellulose and polysulfonemembranes, particularly spiral membranes with transverse flow are used.Such membranes are known and are marketed, for example, by the companyAmicon under the designation "S1Y30". Preferably this firstultrafiltration step is conducted several times in the process accordingto the invention, but particularly at least three times, andappropriately at least five times. It has proven appropriate accordingto the invention to validate the filtration membranes used for thisaccording to the process described in WO 91/12,027 and to check theintegrity of the membrane according to WO 93/02,714. In this way, afirst ultrafiltrate is obtained, which contains effective thymus factorsfor medical and biological purposes.

The second ultrafiltration conducted with this ultrafiltrate on themembrane with an exclusion volume of 3 kD is also preferably conductedseveral times, but particularly at least three times, and preferably atleast five times. Preferred ultrafiltration membranes for this secondfiltration step are, for example, commercially obtainable from thecompany Amicon under the designation S1Y3. It has also provenappropriate to conduct this latter ultrafiltration step as a so-calleddiafiltration, i.e., as a repeated filtration, whereby, analogously to adialysis, undesired components are washed out by multiple filtrationwith the filtrate solution through the filter. In this way it ispossible, without anything further, to separate substances entrainedwith the extraction solution, such as, for example, buffer salts, and toreplace by other additives. It is preferred according to the inventionto dilute the retentate concentrated by means of the secondultrafiltration on the 3-kD membrane with a mannitol solution and torepeat the filtration. In this way, the extraction solution can berebuffered in a simple way. The retentate obtained in this way accordingto the invention can be lyophilized without anything further whileretaining its biological activity.

The invention also concerns a pharmaceutical composition obtained bymeans of the method described above as well as its use for theproduction of a pharmaceutical with immunomodulating andanti-inflammatory action, which is stable during storage.

The invention will be explained in more detail on the basis of thefollowing examples.

EXAMPLE 1

A thymus extract, which is commercially obtainable from sanorell pharmaGmbH & Co., Rechtmurgstrasse 27, Baiersbronn, Germany under thedesignation Thymosand®, was used in the following and is denoted belowalso as UF5. This thymus extract was already ultrafiltered or purifiedfive times by the manufacturer, after removal of the solids, through avalidated membrane described in WO 91/12,027 (UF5). This commerciallyobtainable preparation was then concentrated 10 times (200ml) by meansof a concentrator CH2 (obtainable from Amicon, P.O. Box 1103, Witten,Germany), comprising a supply vessel, a hose pump and an ultrafiltermount over an ultrafilter with Delrin head pieces obtained from Amiconunder the designation S1Y3. The concentrate obtained in this way wasthen diluted to five times the volume by means of a 5% mannitol solutionand then concentrated again to the original volume. This process wasrepeated in all five times. The ultrafiltrate was discarded and theretentate containing the thymus peptides in high concentration wassubjected to a sterile filtration (0.2 μm) and then lyophilized. Thethus-obtained lyophilizate is designated in the following as TML and, incontrast to the initial product, was shown to be extraordinarily stableduring storage for several years even at room temperature.

EXAMPLE 2

Solutions with a protein concentration (Lowry method) of 800 μg/ml wereprepared from the TML lyophilizate obtained in Example 1 and theirbiological activity was measured on blood cells according to the processof Hartung et al. (Hartung, T., A. Sauer and A. Wendel, Biochem.Pharmakol. Univ. Konstanz: W. Cytokine response of whole blood; 25^(th)Annual Conference of the Society for Immunology, September 1994;poster). Thus the cytokine release from lymphocytes in batches of wholeblood cultures is measured after stimulation with phytohemagglutinin(PHA).

The assay described in this report was conducted with PHA and TML orpermeate as costimulators as well as the corresponding negative orpositive controls. The cytokines IL-1β, IL-2, IL-6, IL-10; TNF-α andIFN-γ were measured.

Materials and Methods

Blood Collecting: Citrated Blood (Monovette, Sarstedt)

Culture batches: 4-well culture dishes (4×1 ml) with cover (Nunc; Art.No.: 176740)

The culture batches are prepared at the latest 4 hours after bloodcollecting.

Processing: Specimen Tubes (Sarstedt, Art. No.: 60036550), tablecentrifuge EBA 3S (Hettich)

Cytokine Measurement: Quantikine Kits (Elisa of R&D Systems GmbH,Borsigstr. 7, 65205 Wiesbaden)

Microplate Photometer HTIII (Anthos)

Production of RPMI medium (under laminar flow): 900 ml of water forinjection are placed in a sterilized 2-liter Erlenmeyer flask and apackage of powder-form RPMI-1640 (AutoMod, Sigma; Art. No.: R7755) isintroduced while stirring. Then 10 ml of a 200 mM L-glutamine solution(Sigma Art. No.: 6-7513) are added.

238.3 mg of HEPES (Sigma; Art. No.: H-0878) are weighed out in asterilized small glass beaker, dissolved in a small amount of water andadded to the RPMI solution. The clear RPMI solution is placed in asterilized measuring cylinder, which is filled to 1000 ml with water forinjection while flushing the RPMI vessel, the small glass beaker and theErlenmeyer flask. The finished solution is stored in a sterile 1-literbottle at +4° C. and can be kept for 1 month.

Preparation of RMPI medium+PHA (under laminar flow): 1.875 mg of PHA(Sigma; Art. No.: L9132) is weighed out in a sterilized 100-ml measuringflask and filled with RPMI medium up to the calibration mark. Thesolution is stirred thoroughly for a short time, divided into 30-mlaliquots in sterilized 30-ml vials provided with sterile stoppers andsealing pieces. Removal is conducted with sterile disposable syringes orcannula.

Preparing the whole-blood batches under laminar flow: 4-well culturedishes are used. The final concentrations are the following: PHA, 15μg/ml, TML 806 μg/ml and UF5/Thymosand®, 50 μg/ml. The culture batchesindividually are comprised of the following components (relative to 1well of the culture dish), which are pipetted directly into the wells ofthe culture dishes.

    ______________________________________                                        Negative controls -                                                                         200 μl of blood, 522 μl of medium, 278 μl of                         PBS or mannitol                                                 Positive controls -                                                                         200 μl of blood, 522 μl of medium + PHA,                                278 μl of mannitol solution ("stimulates/                                  mannitol volumetric equilibration")                             TML sample -  200 μl of blood, 522 μl of medium + PHA,                                278 μl of TML ("stimulates + TML")                           Thymosand ® specimen -                                                                  200 μl of blood, 522 μl of medium + PHA,                                278 μl of Thymosand ® (lot no. 4019550)                  ______________________________________                                    

The batches are incubated for 24 h in the culture dishes with closedcover in the incubation chamber at +37° C. in an atmosphere containingCO₂.

Working up the Culture Batches:

The blood specimens are centrifuged in order to obtain the serumsupernatants (10 min; 5600 rpm), made up into aliquots in marked sampletubes of up to 410 μl, each and stored at -70° C.

Conducting the Cytokine Determination

The cytokines IL-1β, 1L-2, IL-6, IL-10, TNF-α and IFN-γ were determinedaccording to the test instructions of the manufacturer R&D Systems, fromthe serum supernatants. Double determinations were conducted for eachmeasurement. In order to obtain statistically valid values, 3determinations were made for each cytokine, each time with a newstandard series, so that 6 measurement values resulted for each sampleper cytokine (with the exception of IL-2).

The results can be derived from the accompanying FIGS. 1-10.

Phytohemagglutinin is used as a synthetic agent, which simulates a feverreaction in cell cultures. As is visible from the figures, the presenceof phytohemagglutinin in all cases causes an increase of the cytokinelevel. The retentate TML obtained according to the invention produces apronounced increase of interleukin-10 production (IL-10) on lymphocytesin the PHA-stimulated cell culture (FIG. 2), which corresponds to aninflammation-inhibiting effect, since IL-10 inhibits prostaglandin-E2synthesis. In contrast to this, the formation of proinflammatoryinterleukin-2 (IL-2) (FIG. 4) as well as also of proinflammatoryIFN-gamma (FIG. 3) is reduced on PHA-stimulated leukocytes. Thecomposition filtered only through a 30-kD membrane, but not through a3-kD membrane (UF5/Thymosand®) causes a reduction of interleukin-10production (FIG. 1). The secretion of TNF-α is increased by the TMLfraction still further to approximately 130% (FIG. 6), whereas theThymosand® fraction that is not free of its low-molecular componentscauses a reduction of TNF-α secretion on PHA-stimulated cells toapproximately 45% (FIG. 5).

The secretion of IL-6 is increased to approximately 230% by TML, whileon the other hand, Thymosand® (UF5) causes a decrease to approximately75% (FIGS. 7 and 8).

The secretion of interleukin-1β is increased to approximately 230% bythe TML fraction (TML retentate) (FIG. 10), while the IL-1β secretion,in contrast, decreases to 60% with the UF5 preparation (Thymosand®)(FIG. 9).

This result is rather surprising, since the quantity ratios of theindividual peptide fractions or thymus factors in the TML do not differin comparison to the Thymosand® initial product.

A composition is obtained with the process according to the invention,which remains stable for years, even at room temperature, and can beprepared with high concentrations (up to 100 times those of Thymosand®).

What is claimed is:
 1. Process for the production of a pharmaceuticalcomposition of thymus factors with immunomodulating andinflammation-inhibiting properties comprising the extraction of ahomogenized thymus tissue by means of an aqueous solution, separatingthe solid components of the extract, and at least one firstultrafiltration of the extract through a filter membrane with anexclusion volume of 30 kD in order to obtain a first ultrafiltrate, saidfirst ultrafiltrate being subjected to at least one additional secondfiltration through an ultrafilter with an exclusion volume of 3 kD toform a retentate.
 2. Process according to claim 1, further characterizedin that the retentate is mixed with an aqueous mannitol solution and isdiafiltered at least once on an ultrafilter with an exclusion volume of3 kD.
 3. Process according to claim 2, further characterized in that thediafiltration is conducted three to seven times with mannitol. 4.Process according to claim 1 or 2, further characterized in that thefirst ultrafiltration is conducted three to seven times on the filterwith the exclusion volume of 30 kD.
 5. Process according to claim 4,wherein the diafiltration is conducted three to seven times withmannitol.
 6. Process according to claim 4, wherein said solid componentsare removed by means of a filter with pore size 1 to 3 μm.
 7. Processaccording to claim 4, wherein the filter for the first ultrafiltrationis validated by means of the Leviviridae virus.
 8. Process according toclaim 4, wherein there is only one second filtration, and wherein saidretentate obtained in said one second ultrafiltration is lyophilized. 9.Process according to claim 1 or 2, wherein said solid components areremoved by means of a filter with pore size 1 to 3 μm.
 10. Processaccording to claim 1 or 2, wherein the filter for the firstultrafiltration is validated by means of the Leviviridae virus. 11.Process according to claim 1 or 2, wherein there is only one secondfiltration, and wherein said retentate obtained in said one secondultrafiltration is lyophilized.
 12. Pharmaceutical preparationcomprising said retentate prepared by the process of claim 1 or
 2. 13.Process according to claim 1 or 2, wherein there are a plurality ofsecond filtrations, and wherein said retentate obtained after completionof said plurality is lyophilized.
 14. A method of treating awarm-blooded animal having a condition requiring immunomodulating actionor anti-inflammatory action, comprising administering to saidwarm-blooded animal an effective amount of a pharmaceutical compositionof thymus factors, said pharmaceutical composition being prepared by theextraction of a homogenized thymus tissue by means of an aqueoussolution, separating the solid components of the extract, and at leastone first ultrafiltration of the extract through a filter membrane withan exclusion volume of 30 kD in order to obtain a first ultrafiltrate,wherein the first ultrafiltrate is subjected to at least one additionalsecond filtration through an ultrafilter with an exclusion volume of 3kD to obtain retentate comprising said thymus factors.